In the life sciences, isolation of specific biomolecules of interest from complex mixtures and assays to identify those molecules and their interacting partners are commonplace. Such methods tend to be performed on solid phase substrates, normally made of glass, silica, or plastics, such as polypropylene and polystyrene, and to increase throughput and improve efficiency, these substrates are typically used in the form of small beads, columns, microscope slides, multi-well plates or membranes. The basic assumption has always been that the surface characteristics of the substrate does not seriously affect the various interactions that are required to take place during the screening or separation process. However, this is not necessarily the case and the lack of suitable solid phase substrates has lead to non-optimal processes and, in some cases, failure of the processes to work at all. For example, in immunoassays, it is well known that immobilization or coupling of peptide or protein antigens on plastic substrates such as latex beads and polystyrene multi-well plates can lead to conformational changes in the antigen resulting in poorer than expected binding with the antibody and sometimes complete failure in the assay (Kabat, E. A., Basic principles of antigen-antibody reactions, in Methods of Enzymology, Vol. 70, Colowick, S. P., and Kaplan, N. O., Eds., Academic Press., New York, 1980, P3; Dierks, S. E., Butler, J. E., and Richerson, H. B., Altered recognition of surface adsorbed compared to antigen bound antibodies in the ELISA, Mol. Immunol., 23, 404, 1986) The opposite situation also exists in that a weak intrinsic interaction between an immobilised antigen and antibody may be converted into a strong interaction in a ternary complex of antigen, antibody and solid phase matrix (Stevens, F. J., “Considerations of the interpretation of the specificity of monoclonal antibodies determined in solid phase immunoassays (Chapter 13, P239, last paragraph in CRC Immunochemistry of Solid Phase Immunoassays, 1991). Between the two extremes of no/poor binding when there is supposed to be high binding affinity and high affinity binding when the reality is low binding affinity (false positives in solid phase screening methods) there is an enormous variation of outcomes that are dependent on the biomolecule being immobilised and the solid phase material. The kinetics of solid phase interactions differ significantly from classical liquid phase interactions and there is no rational process to relate the two interactions when the influence of the solid support becomes significant (see for example Kabat, E. A., Basic principles of antigen-antibody reactions, in Methods of Enzymology, Vol. 70, Colowick, S. P., and Kaplan, N. O., Eds., Academic Press., New York, 1980, P3; Karush, F., The affinity of antibody: range, variability and the role of multivalence, in Comprehensive Immunology, Vol 5, Litman, G. W. and Good R. A., Eds., Plenum Press, New York, 1978, P85; Franz B. and Stegemann, M., The kinetics of solid phase microtiter immunoassays, CRC, 1991, Ch. 18, P277)
Rather than solve this challenging problem of solid phase effects, current strategies normally try to avoid the issue altogether. A common strategy has been to identify surface coatings with minimal “non-specific” binding but with the potential to covalently bind capture agents that are subsequently used to capture their complementary binding molecules.
For example, an important category of capture agents for separations and assays are antibodies and there are a number of methods to immobilize antibodies onto a substrate (see Ed Harlow and David Lane; Antibodies: A laboratory manual by Cold Spring Laboratory, (1988)). Covalent attachment of antibodies to a solid substrate surface can be categorized into three broad classes, as follows.
In the first class, protein A or protein G is first covalently attached to a substrate to act as a capture molecule for the antibody. The antibody requires this capture molecule to bind it to the substrate surface and this interaction is stabilized by cross-linking with a bifunctional coupling reagent such as dimethylpimelimidate (DMP). As both protein A and protein G bind to the Fc region of the antibody, the antigen binding site of the bound antibody will be oriented correctly for optimal subsequent interaction with antigens. This technique tends to be expensive and initial coupling of protein A or protein G onto the solid support is random, leading to uncontrolled orientation and non-optimal antibody loading due to a limited number of protein A or G molecules being bound to the substrate in an orientation which is suitable for antibody binding.
A second type of coupling method uses substrate surface coatings having reactive groups that directly couple certain amino acid side chains in the antibody such as lysine. The main disadvantage of this approach is the lack of control on which lysine(s) in the antibody is/are coupled to the substrate surface. Poor orientation and damage to the antibody are likely outcomes.
A third technique involves activating the antibody first and then coupling the antibody onto a substrate having some reactive groups on its surface. This technique has the same disadvantages as the previous method except when periodate is used to activate the antibody. The periodate breaks the sugar rings in the Fc region and allows the antibodies to be coupled to the substrate bound reactive groups such as amines. In this case, orientation of the antibody can be controlled.
In the prior art, there are many examples on the use of small molecule ligands to bind or capture proteins. For example, the strong binding affinity of biotin to streptavidin can be used. However, if biotin is coupled onto the substrate surface then the antibody needs to be fused or coupled to the streptavidin sequence which greatly complicates the process.
As another example, glycogen synthase kinase-3 (GSK-3) inhibitors have been coupled onto substrate surfaces to identify their actual intracellular targets (Knockaert et al., Identification following affinity purification on immobilised inhibitor, J. Biol. Chem. 2002 277:25493-25501). As another example, Schreiber et al. have synthesized a library of over 2 million unique chemical compounds on small latex beads to screen against cells and multiple proteins. The researchers also claimed printing such compounds on to glass slides, creating small molecule microarrays to probe potential protein targets (Target-oriented and diversity-oriented organic synthesis in drug discover; Science 2000, 1964-1969). However, the experience of most laboratories is that the ligands identified from screening some kind of library invariably binds to native interaction regions of the target protein. If the objective is to orient such interaction regions of the target protein, then existing approaches are very limiting.
As well, just because a protein is bound to the substrate surface through some small molecule ligand does not necessarily mean that the protein will remain in its preferred orientation and conformation. As mentioned before, non-optimal surfaces can lead to conformational changes in the protein resulting in poorer than expected signal to noise ratio and sometimes complete failure in the assays.
Whether by covalent coupling or non-covalent immobilization, there is a need to develop synthetic surface coatings that stabilize and maintain a biological molecules such as antibodies in some preferred orientation. The use of another biological molecule (e.g. protein A) to orient the target molecule (e.g. antibody) only shifts the problem.
There are now a number of highly parallel or combinatorial processes that can potentially generate millions of ligands and identify potentially useful leads. The earliest version of such concepts is that of Mario Geysen who describes methodologies to identify peptide ligands using antibodies to “select” from a large number of peptides, those peptides which bound the antibody. One version was an incremental strategy where a set of 400 dipeptides were immobilised on individual solid supports and tested for their binding affinity. The leads were progressively “built up” to identify higher binding ligands of tri-, tetra- and longer peptides until the appropriate degree of complementarity as judged from the binding characteristics was achieved (see Geysen, H. M., Rodda, S. J. and Mason, T. J., A priori delineation of a peptide which mimics a discontinuous antigenic determinant, Mol. Immunology, 23, 709-715, 1986; Geysen, H. M., Antigen-antibody interactions at the molecular level: adventures in peptide synthesis, Immunology Today, 6 1985, 364-9). In this case, good binding characteristics on the solid phase did not necessarily mean that there was good binding of the peptide ligand in solution or good binding when the ligand was transferred to another solid phase. The surface factors were not part of the discovery process.
More recently, published International patent application WO 03/095494 describes a way of assembling a large library of molecular coatings. In brief, this application describes polymeric surface coatings of the formula B-S-F, where B is a copolymer of at least one passive constituent P and at least one active constituent A, S is a spacer unit and F is a chemical or biological functional group, wherein S is attached to the active constituent A of copolymer B, and wherein the coating has at least one point of diversity selected from P, A, S and F. The functional group F can be a site for further diversity or a group capable of binding or chemically reacting with some biological molecule.
The ability to generate a vast number of different surface coatings of peptide, small molecule or other ligands does not in itself increase the success rate of identifying useful surfaces. Key solid phase applications not only require surfaces with high and low non-specific binding capabilities but also specific binding characteristics that may preferentially orient a target molecule such that some other part of the molecule is freely accessible for subsequent interaction with its complementary binding molecule. Another example in bio-separations is specific high binding capacity but efficient release under some slightly different conditions, e.g., pH or salt change. To efficiently identify such surface coatings, there must be some design elements to complement the capability to assemble and screen millions of surfaces.
Computational chemistry, which incorporates a variety of different methods developed and applied since the early 1980s, is now a well-established approach to identifying new drug leads in the pharmaceutical industry. The main focus has been on generating methodologies and computer programs to design potential small molecule compounds that would bind into a protein binding site or prevent a protein-protein interaction. Biological and chemical databases, virtual screening, pharmacophore modelling, 3-D molecular modelling, QSAR, structural prediction of homologous proteins, to cite a few, are routinely used techniques and have successfully led to the design of new drugs such as HIV protease inhibitors for treating AIDS (Leon et al.; Approaches to the design of effective HIV-1 protease inhibitors, Curr. Med. Chem., 2000, 7, 455).
At the early stage of drug design, computational chemists improve decision-making and help to accelerate the discovery process by increasing the speed and decreasing the cost of identifying lead compounds. This can be achieved by eliminating unpromising compounds and/or by identifying ones which fulfill some criteria that have been identified as important for biological activity. This virtual screening can be performed mainly by using QSAR type models, pharmacophore models, and/or docking techniques. There are many variations on the theme and the choice of the techniques and all their combinations will mainly depend on the number of candidate molecules to be virtually screened and on the knowledge of the target. In brief, the approach involves selecting compounds that fit a feature, a ligand or a receptor.
Assembling and identifying surfaces with the correct set of functionalities in their correct spatial distribution for any particular target protein is a time consuming process. While computational methods are well established in drug discovery, design of ligands for surface discovery has unique requirements that make the end goals very different. In drug discovery, the focus is on identification of high affinity ligands with “drug-like” characteristics in terms of their oral availability, toxicity, etc. As discussed, the solid phase is effectively a tertiary component of the solid phase assay. Even if the strategy is covalent coupling to immobilise some biomolecule to some surface, the effective binding energies after incubation for 16 hrs can be >80% due to non-covalent surface interactions (see CRC Immunochemistry of Solid Phase Immunoassays, J. E. Butler Ed. Chapter 1, p 11).
Some of the differences between designing surfaces for discovery purposes and computational methods in drug discovery are as follows;                a. The ligands responsible for binding a molecule of interest to a surface are actually part of the surface itself and since they are binding macromolecules that are far larger, the binding contributions of the remaining components of the surface must also be considered in the design process.        b. Unless the biomolecule being immobilised on a surface is a spheroid having uniform surface characteristics, it is unlikely that there is random orientation in respect to a specific solid phase. The opportunity to design ligands that preferentially target certain regions of the target biomolecule also means that the design process can incorporate features that preferentially do not target other regions.        c. It is certain that multiple modes of association exist between the incoming biomolecule, small molecule binding ligands and other surface components. As the ligands are part of a contiguous surface, different pharmacophore ligands targeting different sections of an overall binding site on a biomolecule can be incorporated into the surface without the need to achieve some arbitrarily chosen affinity goal.        
There is an opportunity to design small molecule binding ligands as an integral part of the surface component and additionally tune surface characteristics to an intended application.
To address these challenges, the present invention seeks to provide computer-based methods for designing structural features on an artificial surface to capture and manipulate the orientation of different molecules, such as protein classes, and where the surface coating can preferentially enhance specific orientation of those molecules. With respect to the life sciences, these methods are intended to enable identification of optimised surfaces for new bioassays as well as greatly improve the performance in existing bioassays.